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. 2024 Apr 6;30(4):633–645. doi: 10.1007/s12298-024-01444-7

Genome-wide identification and expression analysis of the ALDH gene family and functional analysis of PaALDH17 in Prunus avium

Sitian Li 1, Xiu Wang 1, Wanxia Wang 1, Zhongxing Zhang 1, Xingbin Wang 1, Qingxia Zhang 2, Yanxiu Wang 1,✉
PMCID: PMC11087402  PMID: 38737320

Abstract

ALDH (Aldehyde dehydrogenase), as an enzyme that encodes the dehydroxidization of aldehydes into corresponding carboxylic acids, played an important role inregulating gene expression in response to many kinds of biotic and abiotic stress, including saline–alkali stress. Saline–alkali stress was a common stress that seriously affected plant growth and productivity. Saline–alkali soil contained the characteristics of high salinity and high pH value, which could cause comprehensive damage such as osmotic stress, ion toxicity, high pH, and HCO3−/CO32− stress. In our study, 18 PaALDH genes were identified in sweet cherry genome, and their gene structures, phylogenetic analysis, chromosome localization, and promoter cis-acting elements were analyzed. Quantitative real-time PCR confirmed that PaALDH17 exhibited the highest expression compared to other members under saline–alkali stress. Subsequently, it was isolated from Prunus avium, and transgenic A. thaliana was successfully obtained. Compared with wild type, transgenic PaALDH17 plants grew better under saline–alkali stress and showed higher chlorophyll content, Superoxide dismutase (SOD), Peroxidase (POD) and Catalase (CAT) enzyme activities, which indicated that they had strong resistance to stress. These results indicated that PaALDH17 improved the resistance of sweet cherries to saline–alkali stress, which in turn improved quality and yields.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-024-01444-7.

Keywords: Sweet cherry, ALDH Gene family, Saline–alkali stress, PaALDH17, Functional characteristics

Introduction

Saline–alkali stress was a common stress that seriously affected plant growth and productivity. Soil salinization, as a major abiotic stress, seriously affected the growth and development of plants worldwide, as well as the yield and quality of economic crops (Chaves et al. 2009). In order to survive, plants had evolved multiple regulatory mechanisms in molecular, cellular, and physiological processes to cope with environmental stress (Zheng et al. 2020). Saline–alkali soils were typically characterized by high salinity and high pH. Saline–alkali soil: the salt content ranges from 1.0 to 2.0%, and the pH value is between 7.0 and 9.0; Severe Saline–alkali soil: Salt content greater than 2.0%, pH value above 8.5. In the natural environment, salt stress was mainly caused by neutral salts (such as NaCl and Na2SO4), while Saline–alkali stress was usually caused by alkaline salts (such as NaHCO3 and Na2CO3) (Fang et al. 2021; Cao et al. 2022).

It is well known that stressful conditions can induce the expression of stress-responsive genes in plants to mitigate damage to plants caused by adversity (Xi et al. 2013; Yu et al. 2013). Under high salt and drought conditions, plants could make the production of intercellular messenger abscisic acid (ABA), which induced the transcription expression of stress-related genes to resist stress (Xu et al. 2022; Gabor Jakab and Mauch-Mani 2005; Vishwakarma et al. 2017). Under Saline–alkali stress, the content of ABA in plants increased by activating the synthesis pathway and inhibiting the degradation pathway of ABA (Yimei et al. 2018). At the same time, Saline–alkali stress led to increased transcription levels and up-regulated expression of genes related to ABA synthesis, resulting in a large amount of ABA synthesis and enabling plants to adapt to the external growth environment (Niu et al. 2018; Cai et al. 2017). Proline protects cell membranes by removing superoxide anions from plants (Razavizadeh and Ehsanpour 2009). Compared with salt or alkali stress, Saline–alkali stress environment has a more serious impact on plants, mainly because under saline–alkali stress, plants need to synthesize a large amount of proline for osmotic regulation and pH regulation (Hailing et al. 2010; Yu et al. 2016), so the expression level of proline is often higher under saline–alkali stress (Zengrong et al. 2013; Ochoa 2008).

ALDH is a protein family that exists in plants, which not only remove excess aldehydes in plants to maintain the balance of aldehydes (Gao and Han 2009), but also improve plant resistance by reducing the toxicity of reactive oxygen species (ROS) in plants (Kirch et al. 2004). Previous studies found that ALDH gene in tomato was significantly up-regulated under saline–alkali stress, and the relative conductivity and malondialdehyde content of transgenic plants were reduced under high salt stress, which proved that the plasma membrane damage of transgenic plants was alleviated (Hailing et al. 2010). Semi-quantitative and real-time quantitative PCR data revealed that a few SiALDH genes (SiALDH7B1, SiALDH12A1 and SiALDH18B2) in foxtail millet were expressed in an organ-specific manner and their expression can be dramatically induced saline–alkali stress, osmotic stress and phytohormone abscisic acid (ABA) (Kotchoni et al. 2006a, b). Furthermore, the transformation of SiALDH2B2, SiALDH10A2, SiALDH5F1, SiALDH22A1, and SiALDH3E2 into Escherichia coli (E.coli) was able to improve their salt tolerance (Zhu et al. 2014). These results showed that transgenic plants containing ALDH gene had strong resistance to damage caused by environmental stress, indicating that ALDH gene improved antioxidant capacity.

Sweet cherry (Prunus avium), as "the first fruit of spring" (Qi et al. 2022), displayed high nutritious and economic value (Ricardo-Rodrigues et al. 2023). But cherries had strict requirements for soil conditions, were not tolerant to flooding, prefer neutral and slightly acidic soil (pH 6.0–7.5), and required cultivation in soil with strong water retention and good ventilation (Yu et al. 2016). Due to unreasonable cultivation of wasteland and development of irrigated agriculture, rainwater impact, and human activities, the salinization degree of soil in various places had been deteriorating, which had seriously hindered the growth and cultivation of sweet cherry, and thus affected the development of sweet cherry economic industry. Previous studies had shown that saline–alkali soil significantly reduced the plant height of sweet cherry AM and BRO varieties by 29.1% and 31.9%, respectively, resulting in a reduction of 15.7% and 47.1% in stem diameter and new shoot number of BRO varieties, and a reduction of 26.6% in new shoot length of AM varieties (Yu et al. 2016). SE Arıcı (2012) studied the response of salt stress treatment to the stems of cherry young trees (Prunus avium × Prunus psudocerasus) in vitro cultured. The results showed that the bud numbers and bud length were significantly increased in the medium supplemented with 0–30 mm NaCl. After testing, the explants cultured on the medium with 50–100 mM NaCl showed symptoms of leaf blade, while the explants grown on the medium with 150 mM showed severe leaf burn symptoms. After measurement, the number of buds, bud length, plant freshness, and leaf chlorophyll content also decreased (Ar et al. 2012). The Food and Agriculture Organization (FAO) reported that due to salt stress, agricultural productivity losses ranged from 20 to 40% annually, totaling $31 million (Vargas et al. 2018).

Additionally, the research on ALDH gene has also made some progress in rice, grape, and other crops. But the sweet cherry ALDH family genes have not been identified and little is known about whether PaALDHS are involved in saline–alkali stress resistance. Therefore, in this study, 18 ALDH genes were identified in the sweet cherry genome. After analyzing the expression level of PaALDH17 gene under saline–alkali stress by qRT-PCR the function of PaALDH17 in A. thaliana was studied, aiming to excavate the excellent resistance genes of sweet cherry and provide a theoretical basis for the study of sweet cherry under saline–alkali stress.

Materials and methods

Plant materials

Prunus avium was subcultured at 30-day intervals on Murashige and Skoog (MS) solid medium containing 30 g/L Sucrose, 8 g/L Agar, 0.5 mg /L 6-BA and 0.1 mg /L NAA at 25 C with a 16 h/8 h light/dark period (pH 5.8–6.0). During subgeneration, the 3-4 cm stem segments of tissue-cultured Prunus avium seedlings were cut in ultra-clean workbench and placed into the subculture medium. The seedlings were allowed to  grow into healthy seedlings for  one month. Prunus avium rooting medium consisted 1/2 MS + 30 g/L Sucrose + 7 g/L Agar + 0.5 mg/L NAA + 0.5 mg/L IBA, pH 5.8–6.0.

The wild-type A. thaliana accession Columbia (Col-0) was preserved and donated by XiaoFei Wang Laboratory of Shandong Agricultural University. A. thaliana solid medium used consisted of  MS + 30 g/L Sucrose + 8 g/L Agar, pH 5.8–6.0. After 2 weeks of growth, A. thaliana was transplanted into plastic pots and cultured in medium (substrate:vermiculite = 3:1) with light 16 h/ dark 8 h, 25 °C.

Treatment of saline–alkali stress of Prunus avium seedlings

When the seedlings grew to eight true leaves, healthy seedlings with consistent growth were selected and moved to a plastic box containing 1/2 Hongland’s solution (pH = 6.8 ± 0.2) (Hu et al. 2018) for 7 days preculture, and then incubated in Hongland’s solution for another 7 days with five plants in each bowl. The oxygen was continuously oxygenated with an electric nutrient solution pump, and the nutrient solution was replaced every 7 days. Plants were grown at 26 °C with a 16 h/8 h light/dark cycle, 6000 lx of light and 85%/80% of relative humidity. After 2 weeks the seedlings were treated with a nutrient solution containing 100 mM 1:1 NaCl: NaHCO3 (pH 8.0). Seedlings grown in nutrient solution served as controls. Three biological replicates were independently performed, and each treatment contained five plants in one biological replicate. Samples were taken at five-time points (0, 6, 12, 24, 48, and 72 h respectively).

Bioinformatics analysis of the ALDH gene family in sweet cherry

AtALDH, MdALDH and PaALDH gene conserved proteins were found from the A. thaliana database (TAIR: https://www.A.thaliana.org/), apple (Malus domestica Borkh.) database (http://www.rosaceae.org/projects/apple-genome), and sweet cherry (Prunus avium) genome (http://www.rosaceae.org/projects/sweetsweetcherry-genome), respectively. The ALDH conserved domain (PF00171) was downloaded from the database (http://pfam.sanger.ac.uk/), and then used as a model to search the sweet cherry protein database by HMMER3.0 software, and the parameters were default values. The candidate sweet cherry ALDH protein family members were obtained and were verified by the online software Pfam (http://pfam.xfam.org/) and SMART (http://smart.embl.de/) to determine whether it has a PF00171 conserved domain, and finally 18 sequences were screened out.

The protein sequence was analyzed using the ExPASy online (http://web.expasy.org/protparam/), and the amino acid length, relative molecular mass, potential of hydrogen (PH) and the average protein hydrophilicity were predicted. Chromosomal localization was visualized using the software TBtools.

The ALDH gene family phylogenetic tree was constructed with MEGA 7.0 software. The structure of sweet cherry ALDH gene was analyzed by TBtools software. The distribution of conserved motifs was analyzed using the MEME online tool (https://meme-suite.org/meme/tools/meme), and the results were visualized by TBtools software to draw a map of the distribution of conserved motifs.

The 2000 bp upstream sequence of the PaALDH members were extracted using TBtools software. The cis-acting elements in the promoter region of the sweet cherry ALDH gene family were analyzed using the PLACE online tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Quantitative real-time PCR

Primers were designed using DNAMAN based on sequences from the sweet cherry genome. Quantitative real-time PCR primers were listed in Table S1. The leaf tissue cDNA of Prunus avium was used as the template for real-time PCR. Quantitative real-time PCR was performed: the RNA extraction kit was purchased from Beijing Huayueyang Biotechnology Co., Ltd. and the reverse transcription kit for the synthesis of the first strand of cDNA was purchased from Takara Bioengineering (Dalian) Co., Ltd. PaRPL13 (Accession number: TC5178) was selected as the internal reference gene for quantitative real-time PCR (Ye et al. 2015). The reaction system was: 10 μL TB Green® Premix Ex Taq™ (Tli RNaseH Plus), 1 μL upstream primers (10 μmol/L), 1 μL downstream primers (10 μmol/L), 2 μL template, and 6 μL ddH2O. Reaction conditions were as follows: pre-denaturation at 95 °C for 3 min, denaturation at 95 °C for 5 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, and repeated for 40 cycles, and total volume was 20 μL. Three replicates were performed for each sample. The data were calculated using 2−ΔΔCt method (Zhu et al. 2021a, b), and the difference was analyzed by Duncan test of single-factor ANOVA (P < 0.05).

Cloning and expression vector construction of PaALDH17

In this study, the expression pattern of PaALDH in sweet cherry leaves under salt stress was analyzed by qRT-PCR and evolutionary tree analysis. The results showed that PaALDH17 had a significant response to saline–alkali stress.

The specific cloning process is as follows: 0.1 g of leaves were measured and total RNA of the samples was extracted by Trizol method (Guo et al. 2020). Reverse transcription was performed according to TAKARA's PrimeScripttMRT Reagent Kit with gDNA Eraser (Perfect Real Time). CDS of PaALDH17 sequences were retrieved from the sweet cherry genome database, and specific primers were designed with DNAMAN software (Table S1). PCR amplification was performed. Reaction procedure: pre-denaturation at 95 °C for 5 min, 95 °C denaturation 30 s, 58 °C annealing 42 s, extension at 72 °C for 100 s, and repeated for 42 cycles. Extension at 72 °C for 10 min. The PCR products were electrophoreted with 1.25% agarose gel, and the target bands were recovered using the SanPrep column DNA gel recovery kit of Shanghai sangon biotech Co., LTD. pMD19-T cloning vector (TaKaRa, Dalian, China) was ligated, transformed into trans5α, and selected positive clones for sequencing. The correctly sequenced PaALDH17 plasmid was extracted, digested with Sma I and Kpn I two restriction enzymes, and ligated to pRI101 expression vector. Finally, it was transformed into E.coli, identified positive single colony, and transformed into Agrobacterium LBA4404 for genetic transformation by freeze–thaw method.

Bioinformatics analysis of PaALDH17 gene

Protein sequences homologous of PaALDH17 were identified in other species using NCBI database. Software MEGA-X was used to construct the phylogenetic tree based on neighborjoining (NJ) (Tamura et al. 2013). The cis-acting elements on PaALDH17 promoters were predicted by PlantCare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The physicochemical properties of the protein was analyzed by ProtParam website (http://web.expasy.org/protparam/). Protein amino acid sequence alignment was performed by DNAMAN software.

Agrobacterium‑mediated transformation of A.thaliana

Based on the method of (Hu et al. 2015), transgenic A. thaliana seeds were obtained by inflorescence infection method. The seeds were first treated with 75% ethanol for 5 min, then with 26% sodium hypochlorite (NaClO) for 10 min, and finally washed with deionized water (ddH2O) for 4–5 times. Then the seeds were seeded on MS medium (containing 30 mg/L Kan), and the resistant plants were screened by PCR to obtain heterozygous transgenic plants. After three successive generations of screening, the homozygous transgenic plants of T3 generation were obtained.

Saline–alkali stress treatment and determination of related indexes in A. thaliana

Seeds of A. thaliana wild-type and T3 generation homozygous transgenic lines are sterilized first, then inoculated on MS medium and MS medium (containing 30 mg/L Kan) for 3 days at 4 °C, respectively. After that, transfer to the incubator to make them grow (light 16 h/8 h dark, 22 °C). After 15 days, they were transplanted into plastic pots, and three transgenic A. thaliana strains and wild-type (WT) were cultured under normal stress and saline–alkali stress (100 mM 1:1 NaCl:NaHCO3, PH = 8.0) for 20 days, and the chlorophyll content, relative conductivity (REC) malondialdehyde content (MDA), proline content (Pro) and activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were determined by saline treatment and phenotype.

Determination of chlorophyll content refer to Cheng (2020). Three biological repeats were set for each of the above treatments. A. thaliana was cut into pieces and treated with 95% ethanol for 12 h. The absorbance was measured at 649 nm and 665 nm with 95% ethanol as blank control. Also, the Pro content was determined by Júnio (2017). The content of MDA was determined by thiobarbituric acid method (Lefèvre et al. 1998). The SOD, POD, and CAT activities were measured on a spectrophotometer using kits from Suzhou Keming Biological Co, Ltd. Relative conductivity were measured by conductivity method (DDS-307).

Statistical analysis

Experimental data were presented as means ± standard error (SE) of three independent replicates. Excel 2010 was used to organize the statistical data, SPSS 22.0 (IBM, Armonk, NY, USA) was used to analyze the data, TBtools was used to draw the hot map, Origin 8.0 was used to make graphs, and Duncan method of single-factor ANOVA was used to compare the significant level of difference (P < 0.05).

Results and analysis

Identification of sweet cherry ALDH gene family

In this study, 18 PaALDH proteins, 15 AtALDH proteins and 39 MdALDH proteins amino acid sequences were used to construct phylogenetic tree, and the genetic relationship was studied (Fig. 1). 72 ALDH genes were divided into 4 subgroups (Fig. 1). Sweet cherry ALDH members mainly were in subgroups 1 and 3, and contained six ALDH proteins, while the ALDH members were least common in subgroups 4, which contained two ALDH member.

Fig. 1.

Fig. 1

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Phylogenetic analysis of ALDH proteins in A. thaliana, apple and sweet cherry. *Blue diamond represents A. thaliana; red circle represents apple; yellow triangle represents sweet cherry

The physicochemical properties of PaALDH proteins showed (Table S2) that the length of the CDS ranged from 1464 to 3339 bp; The number of amino acids was 487 ~ 1112aa; The relative molecular mass was between 53.2 and 122.06 kD; The isoelectric points were between 5.13 and 9.1; The instability index was between 27.57 and 52.9; The fat index was between 75.41 and 104.96, of which CpS0033G884m0 had the largest fat index and CpS0037G61m0 the smallest. In addition, the proteins CpS0033G884m0, CpS0034G820m0, and CpS0041G170m0 with a fat index greater than 100 were hydrophilic proteins, and the remaining 15 proteins were hydrophobic proteins.

The secondary structure of ALDH family proteins shown in Table S3, which displayed that the secondary structure contained mainly Alpha helix, Extended strand and Random coil, among which α-helix was between 26.44 and 43.12% and random coiling was between 38.78 and 58.36%, the extended chain structure ranged from 13.57 to 26.21%, with the largest proportion belongs to Random coil, which may be a unique feature of secondary structure of sweet cherry ALDH family proteins.

Chromosomal location analysis of sweet cherry ALDH family

To determine the chromosomal distribution of sweet cherry ALDH, 18 PaALDH genes were chromosomally mapped (Fig. 2). They were found to be distributed on 8 chromosomes of sweet cherry, of which chromosomes 5 and 7 were the most distributed with 4 genes. There were three genes on chromosome 1 and two genes each on chromosomes 4 and 8; There was one gene on chromosomes 2, 3 and 6, and no PaALDH family gene was found on other chromosomes. In summary, the results showed that 18 PaALDH genes were unevenly distributed on 8 chromosomes.

Fig. 2.

Fig. 2

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The position of sweet cherry ALDH gene family on chromosome. *Each chromosome is divided by 0.1 Mb, and the color change in this region represents the gene density (blue: low; red: high)

Analysis of sweet cherry ALDH gene family structure and conserved domains

A total of 18 sweet cherry PaALDH protein conserved motifs were identified using MEME online software (Fig. S1), which was predicted to contain 10 motifs, and they varied slightly in the number and types of motifs. PaALDH protein of class I included motif 1–10, however, the PaALDH protein in Class II varied in amounts of motifs, with CpS0021G571m0 and CpS0033G884m0 only 5 motifs.

At the same time, the Neighbor-Joining method was used to construct the phylogenetic tree (Fig. S2), which showed that the gene family could be divided into two groups: I, II. The PaALDH gene family contained 11–22 exons and 15–21 introns, which showed the gene structures on most sequences are relatively similar, but the differences between lengths were large, so it was speculated that the ALDH gene family had mutated during the evolution.

Analysis of the cis-acting element of sweet cherry ALDH

To analyze the potential expression regulation mechanism, we analyzed the nucleotide sequence of the upper 2000 bp of the sweet cherry ALDH gene family (Fig. 3). It was found that most cis-acting elements contained various stress elements, including drought (MBS), salicylic acid (TCA), abscisic acid (ABRE) and anaerobic (ARE). Additionally, some cis-acting elements were related to hormone responsive element, with auxin response element (TGA), gibberellin (TATC-box, GARE-motif) and others, which indicated that the function of PaALDH may include a series of biological processes, such as regulating plant growth and development, plant hormone signal transduction, and plant resistance to stress.

Fig. 3.

Fig. 3

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Cis-acting components of sweet cherry ALDH gene family

Prediction of ALDH gene function in sweet cherry

In order to explore the interaction of the proteins encoded by the sweet cherry ALDH gene family, we predicted the interacting proteins of sweet cherry ALDH. The results (Table S4) showed that CpS0037G61m0、CpS00128G65m0 and CpS00128G67m0 may interact with malonate semialdehyde dehydrogenase (acetylation)/methylmalonate semialdehyde dehydrogenase, CpS0034G1771m0 interacted with NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, CpS0019G2437m0、CpS0041G170m0 and CpS0020G876m0 were associated with acetaldehyde dehydrogenase. It is speculated that sweet cherry ALDH may be involved interacting these proteins to participate in plant stress resistance.

Heatmap analysis of PaALDH gene under saline–alkali stress

According to qRT-PCR, 18 PaALDH genes were significantly induced under saline–alkali stress, of which 5 genes were down-regulated and 13 genes were up-regulated. In addition, 44% of genes had their maximum relative expression within 24 h (Fig. 4). The expression of most genes, including PaALDH17, increased in leaves under salt and alkali stress, indicating that PaALDH17 may be involved in the response to salt and alkali stress. Combined with evolutionary tree analysis, we saw that PaALDH17(CpS0041G170m0) belongs to a subgroup with AT4G34240.1(ALDH3I1) and AT1G79440.1(ALDH5F1), and it was speculated that PaALDH17 has certain similar functions. After the elimination of ALDH3I1 gene, the sensitivity of transgenic materials to salt stress increased, indicating that ALDH3I1 has a positive response to salt stress (Kirch et al. 2005). Therefore, whether PaALDH17 has the same function as ALDH3I1 deserves further study, and compared with other up-regulated genes, PaALDH17 gene was easier to obtain specific target fragments.

Fig. 4.

Fig. 4

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Heatmap analysis of PaALDH family genes based on transcriptome data under saline–alkali stress. * Blue range represents down-regulated expression and red range represents up-regulated expression

In summary, PaALDH17 gene was selected for subsequent genetic transformation analysis.

Cloning and expression vector construction of PaALDH17 gene

1482 bp band was obtained by PCR amplification (Fig. S3). The sequences of PaALDH17 protein and PaALDH17 protein from other species were selected and analyzed by multiple sequence comparisons, including Prunus persica, Prunus dulcis, Prunus mume, Pyrus x bretschneideri, Malus domestica, Gossypium hirsutum, Juglans regia, Pistacia vera, Carya illinoinensis and Medicago truncatula. The amino acid sequence was performed by DNAMAN software, and the results showed that the PaALDH17 protein of Prunus avium and other species were relatively conserved at the C-terminal, while there was a certain difference at the N-terminal (Fig. S4a). Also, the phylogenetic tree showed that PaALDH17 was closely related to Prunus dulcis (XP 034207422.1) (Fig. S4b).

Screening and identification of PaALDH17-OE A. thaliana

As can be seen from Fig. 5, the PaALDH17 gene was identified at the RNA and DNA levels. At the RNA level, the expression of transgenic plants was significantly higher than WT. Meanwhile, at the DNA level, there were bands between WT and PCR product fragments of transgenic materials with 18S as an internal parameter, while the primers were amplified by PRI101-PaALDH17, the PCR product bands of transgenic materials were clear and there were no bands in WT, indicating that the PRI101-PaALDH17 vector was successfully transformed.

Fig. 5.

Fig. 5

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Expression level of PaALDH17 in A. thaliana. Expression of PaALDH17 in WT was set to “1”. Data are means of three replicates with SE. DNA level identification of A. thaliana. Values not followed by the same letter indicate significant differences between treatments, according to Duncan method of single-factor ANOVA (P < 0.05)

Tolerance of PaALDH17-OE A. thaliana to saline–alkali stress

Salinity and alkali stress will cause more severe chlorosis and wilting of plant leaves. As shown in the Fig. 6, the transgenic plant and the WT plant grew well under normal conditions, with the leaves green and not wilted. However, under saline stress, WT showed more severe wilting and yellowing than transgenic plants (Fig. 6). Subsequently, this was confirmed by the determination of chlorophyll content, which was higher in the transgenic plant than WT (Fig. 7).

Fig. 6.

Fig. 6

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The phenotypes of PaALDH17-OE and WT A. thaliana under normal conditions and saline–alkali stress. a The phenotypes of OE-1, OE-2, OE-3 and WT A. thaliana under normal conditions. b The phenotypes of OE-1, OE-2, OE-3 and WT A. thaliana under saline–alkali stress

Fig. 7.

Fig. 7

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Chlorophyll content of PaALDH17-OE and WT A. thaliana under normal conditions (CK) and saline–alkali stress (T)

Under saline–alkali treatment, the three PaALDH17-OE lines also exhibited lower REC and MDA, and higher Pro compared to WT, but were not different between PaALDH17-OE and WT under normal conditions (Fig. 8). SOD, CAT and POD played a great role in plants, helping to clear ROS and reduce the degree of membrane damage (Gill and Tuteja. 2010). SOD, POD and CAT activity in transgenic and WT A. thaliana were measured (Fig. 9). Transgenic A. thaliana had much higher values than the WT control for all measurements (SOD, POD and CAT) under saline–alkali stress. But under normal conditions, there was no difference between PaALDH17-OE and WT. These results suggested that the heterotopic expression of PaALDH17 in A. thaliana enhances its tolerance to saline–alkali stress.

Fig. 8.

Fig. 8

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The REC, MDA, and Pro content of PaALDH17-OE and wild-type (WT) A. thaliana under normal conditions and saline–alkali stress. a The REC. b The MDA content. c The Pro content. Data are means of three replicates with SE. Values not followed by the same letter indicate significant differences between treatments, according to Duncan method of single-factor ANOVA (P < 0.05)

Fig. 9.

Fig. 9

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The SOD, POD, and CAT content of PaALDH17-OE and wild-type (WT) A. thaliana under normal conditions and saline–alkali stress. a The SOD content. b The POD content. c The CAT content. Data are means of three replicates with SE. Values not followed by the same letter indicate significant differences between treatments, according to Duncan method of single-factor ANOVA (P < 0.05)

Discussion

Identification of the ALDH gene family in Prunus avium

Aldehyde dehydrogenases (ALDHs) are the key enzymes that maintain the stability of aldehydes in plants (Tola et al. 2020), which can further maintain the normal growth and development of plants by removing excess aldehydes and reducing the toxicity of aldehydes accumulation to cells under saline–alkali stress conditions (Dash et al. 2019). As a gene involved in the formation of aldehyde dehydrogenases (ALDHs), ALDH plays an important role in abiotic stress responses (Maninang et al. 2015). Saline–alkali stress are the important abiotic stress factors affecting crop growth, development and yield (Mehari et al. 2021), and further causing irreversible harm to plant growth by affecting a series of physiological metabolism and morphological structures of plants (Li et al. 2017; Zhu et al. 2021a, b). In recent years, genes encoding aldehyde dehydrogenases (ALDHs) had been studied in grapes, rice, tomato and A. thaliana (Du et al. 2022), and most ALDH gene family members of different crops were found expressing high under saline–alkali stress, indicating that they can effectively alleviate the damage to plants due to saline–alkali stress, which is consistent with the conclusions of this study also (Raza et al. 2022; Zhao et al. 2017). In addition, the maize genome contained 28 ALDH genes, and the transcription level of ZmALDH was inhibited by drought stress but strongly induced by salt stress (Jimenez-Lopez et al. 2016; Zhou et al. 2012). Therefore, we conducted a study on the ALDH gene of sweet cherries under salt alkali stress. Through qRT-PCR verification, we found that most members of the ALDH gene family of sweet cherries exhibited the same high expression under salt alkali stress, which was consistent with the conclusions of previous studies. These research results indicated that the ALDH family plays an important role in plant resistance to salt alkali stress.

In this study, 18 members of the sweet cherry ALDH gene family were systematically identified, and the biological information of the members was analyzed. The results showed that most of the PaALDH proteins were relatively stable. In addition, subcellular localization results showed that members of the PaALDH family were distributed in chloroplasts, mitochondria, peroxisomes, and cytoplasm, which was similar to ALDH in other species. For example, AtALDH3I1, AtALDH7B4, and OsALDH2B1 were located in chloroplasts, cytoplasm (Kotchoni et al. 2006a, b), and nuclear compartments (Ke et al. 2020), respectively, and were also consistent with members of the tomato ALDH gene family (Jose et al. 2016). In the analysis of the phylogenetic relationship, the evolutionary tree can be divided into four groups. PaALDH was all distributed in each group, indicating that the PaALDH gene is relatively conservative in the evolutionary process, and it is speculated that they possessed similar functions, which lays the foundation for later gene function research. Conserved motif analysis confirmed PaALDH protein of class I included motif 1–10, while the PaALDH protein in Class II varied in amounts of motifs, with CpS0021G571m0 and CpS0033G884m0 only 5 motifs. In this study, cis-acting element analysis displayed that ABRE and ARE elements widely existed, so it was speculated that they play an important role in the induction of sweet cherry ALDH gene expression.

Overexpression of PaALDH17 enhanced tolerance to saline–alkali stress in transgenic A. thaliana

In this study, the expression of PaALDH in sweet cherry leaves under saline–alkali stress was analyzed by qRT-PCR and evolutionary tree analysis. Under salinity stress, PaALDH7/8/10/11/12/14/17/18 gene expression quantity changed significantly within 24 h. According to the evolutionary tree analysis, PaALDH17 and ATALDH3I1 which were sensitive to saline–alkali stress, belong to the same subfamily, so we guessed that PaALDH17 may also be sensitive to salt stress, and the qRT-PCR verification results also indicated that PaALDH17 has high expression level under salt and alkali stress. After gene cloning, we selected PaALDH17 for further study.

On the other hand, we obtained transgenic A. thaliana with PaALDH17. Phenotypic analysis showed that the wild type line undergoes more severe chlorosis and wilting under saline–alkali stress than the transgenic lines, and the measurement results of chlorophyll content also confirm this phenomenon.

Chlorophyll was the pigment responsible for photosynthesis. In this experiment, we measured the chlorophyll content of transgenic A. thaliana and WT A. thaliana and found that the chlorophyll content of transgenic A. thaliana was significantly higher than that of the WT under saline–alkali stress indicated that the photosynthetic system of WT plants was more sensitive to saline–alkali stress.

The change in MDA content reflected the severity of membrane peroxidation in plant cells and the strength of plant response to stress conditions (Aghdam et al. 2012). Thus, the stress tolerance of plants can be inferred by detecting the degree of damage to the membrane system by MDA content (Neill et al. 2002; Tang et al. 2013; Wang et al. 2010). Under saline–alkali stress conditions, the REC and MDA content of PaALDH17 transgenic lines were low (Fig. 7a and c), which was consistent with the study of ALDH in tomato, where the REC and MDA content of transgenic plants were reduced under saline–alkali stress compared to WT plants, demonstrating a reduction in plasma membrane damage in transgenic tomato plants (Hailing et al. 2010). Kotchoni et al. 2006a, b found that ectopic expression of ALDH3I1 and ALDH7B4 significantly reduced lipid peroxidation and malondialdehyde (MDA) levels and increased tolerance to salt stress in transgenic Arabidopsis thaliana. Moreover, over-expression of VvALDH2B4 in A. thaliana enhanced protection against high salt and pathogenic bacteria and resulted in lower MDA levels (Zhang et al. 2012). Furthermore, compared with control plants, the transgenic plants displayed higher germination ratios, root lengths, proline accumulation and antioxidant enzyme activities as well as lower MDA contents when ScALDH21 was introduced into tobacco (Yang et al. 2015). We found that the MDA content of the transgenic plants was lower under salt alkali stress compared to the WT plants. These results showed that the degree of membrane damage in transgenic plants was lower than that in wild-type plants, and transgenic plants containing ALDH gene had strong resistance to damage caused by environmental stress. These results showed that transgenic plants containing ALDH gene had strong resistance to damage caused by environmental stress.

Osmotic adjustments, by accumulating osmoprotectants inside the cell, were essential for reducing the cellular osmotic potential against an osmotic gradient between root cells and the outside saline solution, which eventually restored the water uptake into roots during salinity stress (Golldack et al. 2014). Overexpressing ScALDH21 tobacco plants showed higher growth ratio, higher proline accumulation, and stronger photosynthetic capacities, when subjected to salt stress (Yang et al. 2015). The free proline levels were higher during drought and salt stress conditions and the free proline levels in CsALDH-overexpressing transgenic plants were higher than those in WT plants. Free proline played an important role in effectively mitigating osmotic damage when plants are stressed (Xi et al. 2013). Proline and soluble sugars were usually osmotic protectants that enabled plants to withstand stress. In this study, we examined the contents of the osmoprotectant proline in plants, showing that the Pro content of PaALDH17-OE plants was significantly higher than that of the wild type (Fig. 7b). It is demonstrated that transgenic strains can enhance plant resistance by accumulating osmoregulatory substances proline, thereby making them less harmable. Therefore, the increased accumulation of proline contributed to the increased saline–alkali stress tolerance of transgenic plants.

SOD, POD, and CAT were common protective enzymes in plant tissues that can remove H2O2 produced during metabolism and avoid oxidative damage to cells by H2O2 accumulation (Mittler 2002). In this study, the activity of the three enzymes in the PaALDH17-OE line was significantly higher than that of the wild type (Fig. 8). In this study, consistent with the results obtained from previous studies on abiotic stress response genes, GhSOS1 gene isolated from cotton was transferred into Arabidopsis Thaliana, and it was found that H2O2 content of transgenic plants was significantly reduced, and POD, SOD and CAT activities were higher than those of wild Arabidopsis Thaliana (Chen et al. 2017). Transgenic plants over-expressing VpALDH2B4 showed higher salt-stress induced SOD activity (Wen et al. 2012). These results suggested that transgenic A. thaliana had stronger ability to mobilize protection systems than the WT and had strong ability to scavenge ROS, thus maintaining stability of the membrane system (Halliwell and Barry 2006). In conclusion, these results suggested that overexpression of PaALDH17 gene enhances plant resistance to saline–alkali stress.

Conclusion

In this study, 18 ALDH genes were identified from the sweet cherry genome, and bioinformatics analysis showed that PaALDH was divided into 2 distinct subclasses. Under salt alkali stress, PaALDH17 showed a high expression level. Subsequently, it was found that transgenic strains could protect plants from damage by accumulating more proline, increasing chlorophyll content, and improving antioxidant enzyme activity (SOD, POD, CAT) to deal with saline–alkali stress, which provided a theoretical basis for further studies on the mechanism of saline–alkali stress and molecular breeding of sweet cherry.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 25 KB) (21.3KB, docx)
Supplementary file2 (DOCX 1066 KB) (1MB, docx)

Acknowledgements

This work was supported by National Natural Science Foundation of China (Project No. 31960581) and Gansu Provincial Department of Science and Technology Technology Innovation Guidance Program Project (Project No. 21CX6NM108).

Author contributions

STL and YXW designed the research. STL, XW, WXW performed the experiments. YXW and ZXZ performed the data analysis and interpretation. STL and WXB prepared the figures and tables. STL wrote the manuscript. All authors read, commented on and approved the manuscript.

Data availability

We declare that the submitted manuscript is our work that has not been published before and is not currently being considered elsewhere.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

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Supplementary Materials

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Data Availability Statement

We declare that the submitted manuscript is our work that has not been published before and is not currently being considered elsewhere.

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